The present invention relates to a method and kit for detecting total 1,25-dihydroxy-vitamin D in a biological fluid sample, such as whole blood, plasma, serum, or urine sample.
More in particular, the present invention relates to an immunoassay method and kit, as well as to the related antibodies, suitable for detecting total 1,25-dihydroxyvitamin D in a biological fluid sample which may contain 1,25-dihydroxyvitamin D together with other non active forms of vitamin D, such as 25-hydroxyvitamin D.
Vitamin D is a steroid hormone which plays a fundamental role in skeletal metabolism and calcium homeostasis. In humans and animals, the major forms of vitamin D are vitamin D3 (cholecalciferol) and vitamin D2 (ergocalciferol). Vitamin D3 is primarily synthesized in the skin from 7-dehydrocholesterol in response to exposure to solar ultraviolet-B (UVB), but vitamin intake can also occur from dietary sources such as oily fish, i.e. salmon and mackerel. Vitamin D2 is primarily acquired in the diet from fungal and vegetable sources as well as from supplementation (e.g. Drisdol™ or Sterogyl 15 “A”).
Irrespective of the source, the conversion of vitamins D2 and D3 into a bioactive compound requires two separate hydroxylation steps. In the liver, the enzyme 25-hydroxylase converts vitamin D to 25-hydroxyvitamin D (hereinafter designated as “25(OH)D”). This intermediary metabolite is the major circulating form of the hormone and serves as a reservoir for further hydroxylation to the biologically active metabolite 1,25-dihydroxyvitamin D (hereinafter designated as “1,25(OH)2D”).
The latter step takes place primarily in the renal tubular cells and is catalyzed by the enzyme 1-alpha-hydroxylase. The plasma concentrations of 1,25(OH)2D are highly regulated by a variety of factors, including the serum parathyroid hormone (PTH), and they are normally about 1000-fold lower than the precursor compound 25(OH)D.
Because of their lipophilic nature, the majority of vitamin D and metabolites thereof circulate in the blood-stream bound to the vitamin D binding protein (DBP) (80-90%), also known as Gc-Globulin, and albumin (10-20%). DBP has high affinity for vitamin D metabolites (Ka=5×108M−1 for 25(OH)D and 24,25(OH)2D, 4×107M−1 for 1,25(OH)2D and vitamin D), such that under normal circumstances only approximately 0.03% 25(OH)D and 24,25(OH)2D and approximately 0.4% 1,25(OH)2D are in a free form.
The biological effects of 1,25(OH)2D are mediated primarily by the binding of this bioactive hormone to a specific intracellular Vitamin D Receptor (VDR), which acts primarily by regulating the expression of genes whose promoters contain specific DNA sequences known as Vitamin D Response Elements (VDREs).
The Vitamin D Receptor (VDR) is a ligand-dependent transcriptional regulator belonging to the superfamily of nuclear receptors (NRs). Like the other members of this receptor family, the VDR possesses a modular structure which comprises an amino-terminal A/B domain, a highly conserved DNA-Binding Domain (DBD), a flexible linker region and a C-terminal Ligand-Binding Domain (LBD) which is more variable (Mangelsdorf D J et al., 1995, Cell 83(6):835-9). The C-terminal LBD is a globular multifunctional domain, responsible for hormone binding, dimerization with Retinoid X Receptor (RXR) and interaction with co-repressors and co-activators, which all together are critical for the regulation of transcriptional activities (Haussler M R, et al. 1998, J Bone Miner Res. 13(3):325-49).
The Ligand Binding Domain (LBD) of VDR has been crystallized and its structure solved (Rochel N, Wurtz J M, Mitschler A, Klaholz B, Moras D The crystal structure of the nuclear receptor for vitamin D bound to its natural ligand. Mol Cell 2000; 5:173-179).
The binding of the ligand to the VDR induces a conformational change at the Ligand Binding Domain of the receptor, which in turn increases heterodimerization of VDR with a cofactor, the Retinoid X Receptor (RXR), on a Vitamin D-Responsive Element (VDRE) in the promoter region of the target genes. This in turn leads to opening of the promoter to the transcriptional machinery (Glenville J. et al., 1998 Physiological Reviews 78(4):1193-1231).
Nuclear receptor Ligand Binding Domains (LBDs) are known to have a high content of alpha-helix, which may undergo a large conformational change in response to ligand binding, forming up a hydrophobic pocket. Recently, differences in the conformation of the Rattus norvegicus Ligand-Binding Domain (r-VDR-LBD) when bound to diverse ligands were solved by NMR spectroscopy (Kiran K. Singarapu et al. 2011 Biochemistry 50 (51): 11015-24).
Vitamin D is currently recognized as a pro-hormone which has multiple roles in maintaining optimal health in human beings. It has long been established that marked vitamin D deficiency results in histologically evident bone diseases such as osteomalacia in adults and rickets in children, while vitamin D insufficiency may cause alterations in the parathyroid hormone concentration which, if persisting over time, may contribute to bone loss and fracture. However, although initially identified as a classic regulator of calcium homeostasis, vitamin D is now known to have a broader spectrum of actions, driven by the wide expression and distribution in human tissues of the vitamin D receptor (VDR).
In the last decades, clinical and epidemiological data have provided several evidences that impaired levels of 25(OH)D are associated with an increasing risk of various chronic diseases including cardiovascular diseases, hypertension, myocardial infarction, diabetes, cancer, reduced neuromuscular function, infectious and autoimmune diseases. Even complications of pregnancy such as pre-eclampsia, gestational diabetes, cesarean section, and premature birth might be the tragic sequela of gestational vitamin D deficiencies (Holick M F; 2007 N Engl J Med. 357(3):266-81, Holick M F and Chen T C. 2008 Am J Clin Nutr.; 87(4): 1080S-6S).
However, very few studies have been carried out to associate risks of chronic disease to 1,25(OH)2D levels, due to both complexity and lack of reliability of the measurement methods which are available today.
Therefore, the determination of circulating 1,25(OH)2D, which is the active form of vitamin D, is becoming of increasing relevance in many different clinical applications, either as a diagnostic marker and/or as a therapy monitoring indicator. For instance, the determination of serum 1,25(OH)2D and parathyroid hormone (PTH) levels and a possible correlation thereof may represent an important measure for aiding in the diagnosis of parathyroid diseases as well as for the detection of the onset of secondary hyperparathyroidism in the course of renal failure or the development of vitamin D-resistant rickets (VDRR).
Currently, both in routine clinical and research use there is a wide range of methodologies available for measuring the circulating levels of total 25(OH)D (i.e., 25(OH)D3+25(OH)D2). Commercial, fast, automated chemiluminescence-based immunoassay methods are supplied by Abbott Diagnostics (Abbott Park, Ill., USA, ARCHITECT 25-OH vitamin D assay), DiaSorin Inc. (Stillwater, Minn., USA, LIAISON® 25 OH Vitamin D Total Assay), Immunodiagnostic Systems (Boldon, England, IDS-iSYS 25-Hydroxy Vitamin D (25OHD)), Roche Diagnostics (Mannheim, Germany, Modular Analytics E170 Elecsys® Vitamin D Total assay), and Siemens Healthcare Diagnostics (Tarrytown, N.Y., USA, ADVIA Centaur® Vitamin D Total assay). Besides these assay platforms, there has recently been a steady increase in the use of physical methods based on chromatographic separation followed by non-immunological direct detection (semi-automated liquid chromatography-tandem mass spectrometry, LC-MS/MS), which have been principally developed in specialist laboratories in the United States (e.g. Esoterix Inc. in Calabasas Hills, Calif., Mayo Clinic in Rochester, Minn., ARUP Laboratories in Salt Lake City, Utah and Quest Diagnostics in Lyndhurst, N.J.), Europe (e.g. Ghent University in Ghent, Belgium, and CHU de Liege in Liege, Belgium) and Australia (e.g. Pathology Queensland in Herston Queensland, and Douglass Hanly Moir Pathology in Macquarie Park NSW).
Despite the wide selection of assay platforms for measuring 25(OH)D, there are no automated assay methods currently available for the quantitative determination of the active form of vitamin D in clinical samples. The systemic circulating levels of 1,25(OH)2D are extremely low, in the pg/ml range, and therefore represent a significant bioanalytical challenge for clinical monitoring. Quantitation of 1,25(OH)2D in plasma has been traditionally carried-out by radioimmunoassay (RIA). In order to avoid problems related to handling of radioactivity and the limited shelf-life of radioactive labels, new vitamin D testing methods have recently emerged which mainly rely upon the employment of the LC-MS/MS methodology. However, the reported LC-MS/MS bioanalytical assays for 1,25(OH)2D suffer from the extensive sample preparation procedures or derivatization protocols which need to be carried out in order to achieve the requisite sensitivity and selectivity. At present, the main methods available for the detection of 1,25(OH)2D require performing a number of sample pre-treatment or pre-analytical steps which are usually carried-out manually and may therefore be very time consuming, labor intensive, and expensive.
EP 0 583 945 A discloses an assay for 1,25(OH)2D which involves extracting blood serum using an organic solvent such as ethyl acetate, separating out potentially interfering other vitamin D metabolites using a silica column, and then adding pig receptor protein, radiolabeled 1,25(OH)2D, biotinylated antibody capable of binding to the receptor, and a facilitator protein such as BSA as part of an immunoprecipitation competitive binding assay.
WO/8901631 discloses a competitive binding assay for 1,25(OH)2D (3) which involves adding pig receptor protein, radiolabeled 1,25(OH)2D and biotinylated antibody capable of binding to the receptor to untreated blood serum. The competitive binding assay requires the use of vitamin D transport protein which acts as a screen to minimize interference from related metabolites.
S. SWAMI et al., Bone, Vol. 28, No. 3, March 2001:319-326 discloses an antibody which binds to the hinge portion of the vitamin D receptor (VDR) and which is used in a method for the measurement of VDR. However, such antibody is not able to distinguish between ligand-occupied and -unoccupied VDR and is therefore not useful for the detection of 1,25(OH)2D.
The DiaSorin RIA (Part No. 65100E/100 Tubes; 1,25-Dihydroxyvitamin D) involves the use of organic solvents, extraction instrumentation, and C18-OH columns to separate out potentially interfering vitamin D metabolites such as 24,25(OH)2D, 25,26(OH)2D and 25(OH)D in order to isolate 1,25(OH)2D from the test sample prior to metabolite measurement.
Even the recently commercialized automated assay supplied by Immunodiagnostics for the determination of 1,25(OH)2D (Part No. IS-2400; IDS-iSYS 1,25-Dihydroxyvitamin D) requires a time-consuming and labor intensive sample pre-treatment step which makes use of the IDS proprietary Immunocapsules.
Furthermore, the prior art methods often suffer from limitations in term of assay specificity since cross-reactivity events with other vitamin D metabolites not completely removed from the test specimens during the pre-analytical or sample pre-treatment steps may lead to the measurement of erroneous higher concentrations of 1,25(OH)2D. For example, most immunoassay antibodies significantly cross-react with 25(OH)D, 24,25(OH)2D, and 25,26(OH)2D which may be present in blood at levels 1000-fold greater than 1,25(OH)2D.
There is therefore a strong need to develop an assay method for detecting total 1,25(OH)2D (1,25(OH)2D2+1,25(OH)2D3) which does not suffer from the drawbacks and limitations of the prior art.
In particular, there is a need for an assay method which would enable precise, sensitive and accurate detection of total 1,25(OH)2D (1,25(OH)2D2+1,25(OH)2D3) without requiring time-consuming and labor intensive sample pre-treatment steps and which may possibly be provided in an automated format.
There is also a need for a 1,25(OH)2D assay method which substantially does not cross-react with other vitamin D metabolites which may be present in the test sample.
These and other needs are met by the method, and the related kit and antibodies, as defined in the appended claims, which form an integral part of the description.